The present invention relates to an electrochemical detection method and an apparatus therefor and, more particularly, to an electrochemical detection method and an apparatus therefor, which are used in electrochemical analysis, a chemical sensor, or a biosensor.
An electrochemical detection method is generally used as a method of identifying a material dissolved in the aqueous solution and measuring its amount.
This electrochemical detection method is a method of measuring a current flowing at the electrode in an aqueous sample solution to perform quantitative or qualitative analysis of a material dissolved in the aqueous solution. According to this method, basically, a potential is applied between working and reference electrodes which are immersed in the aqueous sample solution, an oxidation/reduction (redox) reaction of the analyte occurs on the working electrode upon application of the potential, and the magnitude of a current flowing due to this reaction is measured to perform analysis. The electrochemical detection method is commonly used due to its relatively high sensitivity and simpleness.
Typical methods of measuring this current are voltammetry, stripping voltammetry (stripping), and pulse voltammetry methods.
More specifically, the voltammetry method is a method of sweeping the potential of a working electrode immersed in a sample electrolytic solution for measurement with reference to the reference electrode potential, and measuring a current change upon the potential change of the working electrode. When this method is used, the concentration of analyte can be obtained from the measured magnitude of current. In addition, the type of analyte can also be determined from the potential at which the current starts to flow upon potential sweeping. Therefore, both quantitative and qualitative analyses can be simultaneously performed.
However, as this method is performed by sweeping the potential, a charging current flowing in proportion to a potential sweeping rate, electrochemical reactions of coexisting species (e.g., dissolved oxygen and hydrogen ions) except for the current of analyte, and the current caused by the variation of the oxidation states of the electrode surfaces cause noise. As a result, it is difficult to detect an analyte concentration of .mu.mol/l or less.
On the other hand, the stripping method is a method of performing analysis in two stages, i.e., pre-electrolysis and stripping. For example, when metal ions dissolved in a sample electrolytic solution is quantitatively analyzed, a predetermined potential enough to reduce metal ions is applied to a working electrode with reference to the reference electrode potential, thereby depositing a metal on the working electrode in the pre-electrolysis stage. Thereafter, in the stripping stage, the potential applied to the working electrode is swept in a direction to cause oxidation (dissolution) of the metal (analyte). By this potential sweeping, when the potential of the working electrode reaches an redox potential of the metal, the metal deposited on the working electrode is rapidly oxidized and dissolved. At this time, a large current flows at the working electrode and is measured to determine the metal ion concentration dissolved in the sample electrolytic solution. Since this stripping method can achieve a low detection limit, it is mainly applied to analysis of a very small amount of heavy metal ions in water, food, or a body fluid (anodic stripping method). In this stripping method, mercury, carbon, and mercury-modified carbon electrodes are used as the working electrodes.
For example, when a mercury electrode is used as a working electrode for analyzing metal ions in pre-electrolysis, the metal ions are reduced while the potential of the working electrode is kept lower than the reduction potential of metal ions. The reduced metal atoms are reacted with mercury on the working electrode to form an amalgam, thereby preconcentrating the metal atoms on the working electrode. Thereafter, in stripping, when the potential of the working electrode is swept to an oxidation side, the metal preconcentrated on the working electrode (amalgam form) is rapidly oxidized at the redox potential of this metal and dissolved.
At this time, the concentration of metal ions can be determined from the measured current flowing through the working electrode (e.g., DENKI KAGAKU SOKUTEI HOU, Akira Fujishima, Masuo Aizawa, and Toru Inoue, Gihodo Shuppan, PP. 206-208).
By using this method, high sensitivity in the pico-mol region is obtained in analysis of lead, zinc, tin, indium ions, etc.
Two other stripping methods were reported. One is a cathodic stripping method. This method is performed by preconcentrating anions such as chloride, bromide, and iodide onto the working electrode and then stripping these anions by sweeping the working electrode potential to the negative potential region which the reduction reaction of anions take place. The other method is an adsorptive stripping method. This method is performed by preconcentrating an analyte on the working electrode whose surface was modified with material which strongly interacts with the analyte, and then stripping the analyte by potential sweeping.
The pulse voltammetry method is a method in which the potential of a working electrode is swept stepwise, e.g., every several mV or several tens of mV in place of linear sweeping of the working electrode potential, and a current flowing through the working electrode is measured by an electrochemical reaction of the analyte is measured immediately after a charging current flowing upon application of a stepwise voltage is attenuated.
This pulse voltammetry method is used when an analyte which cannot be preconcentrated on an electrode by the stripping method is to be detected at a higher sensitivity than that of the lower sweep voltammetry method. The pulse voltammetry method can achieve a sub-.mu.mol sensitivity, but its sensitivity is lower than that of the stripping by 100 times or more.
As is well known, the high redox cycling of a redox species occurs upon application of different potentials to two adjacent working electrodes in an electrolytic solution, and a current flowing through the working electrodes can be amplified by 40 times or more (J. Electroanal. Chem. Preliminary note, Vol. 267, P. 291, 1989).
If an interdigital electrode is used as these two working electrodes, a measurement can be performed such that one working electrode of the interdigital electrode is swept and the other working electrode of the interdigital electrode is fixed at a constant potential. Therefore, the measurement almost free from the influence of the charging current can be performed.
When different potentials are applied to the two working electrodes in the interdigital electrode, a lower detection limit of 5 to 10 nmol/l can be obtained quantitatively in the analysis such as a metal (Anal. Chem. Vol. 62, P. 447, 1990).
The problems posed by the above conventional techniques can be summarized as follows. It is difficult for the conventional methods described above to detect an analyte which exits in a solution and hardly be deposited on the electrode after the electrochemical reaction (an oxidation/reduction reaction).
In measurement using the voltammetry method which does not include an analyte deposition on a working electrode in pre-electrolysis, a detection limit of 10 nmol/l to 100 nmol/l is obtained even in the pulse voltammetry method.
In this case, even if a small interdigital electrode capable of increasing the sensitivity is used, only a detection limit of about 5 to 10 nmol/l can be obtained.
This detection limit is higher than that of the stripping voltammetry by one or two order of magnitude.
In the conventional stripping voltammetry, this analysis is performed after electrochemically deposited on an analyte such as metal ions, which is dissolved in a sample solution on a working electrode.
For this reason, the analyte whose solubility is not changed by the redox reaction, and exits in the previous state even after the oxidation/reduction cannot be analyzed.
For example, it is difficult to apply this method to electrochemically reversible biological materials such as hydroquinone (p-dioxybenzene C.sub.6 H.sub.6 O), catechol (o-dioxybenzene C.sub.6 H.sub.6 O.sub.2), catechol amine, NADH, or vitamin K.sub.3 (menadione C.sub.11 H.sub.8 O.sub.2), and metal complexes such as ferrocene (C.sub.10 H.sub.10 Fe) derivative, ruthenium hexaamine, or ferrocyanide.
On the other hand, in the case of the adaptive stripping method, it is possible to modify a working electrode with a material which strongly interacts with an analyte and preconcentrates it on the working electrode. Organic molecules may be measured by properly selecting a material (thin film) for modifying the working electrode.
However, a material for modifying the electrode must be selected in accordance with each analyte, and an adsorption state of the analyte in the working electrode is changed in accordance with the ionic strength of the sample electrolytic solution and a slight pH change, thus posing difficult problems for measurement.